Hepatitis C virus is the most common cause of chronic viral hepatitis within the United States, infecting approximately 4 million Americans and responsible for the deaths of 8,000-10,000 persons annually due to progressive hepatic fibrosis leading to cirrhosis and/or the development of hepatocellular carcinoma. Hepatitis C virus is a single stranded, positive-sense RNA virus with a genome length of approximately 9.6 kb. It is currently classified within a separate genus of the flavivirus family, the genus Hepacivirus. The hepatitis C virus genome contains a single large open reading frame (ORF) that follows a 5′ non-translated RNA of approximately 342 bases containing an internal ribosome entry segment (IRES) directing cap-independent initiation of viral translation. The large ORF encodes a polyprotein which undergoes post-translational cleavage, under control of cellular and viral proteinases. This yields a series of structural proteins which include a core or nucleocapsid protein, two envelope glycoproteins, E1 and E2, and at least six nonstructural replicative proteins. These include NS2 (which with the adjacent NS3 sequence demonstrates cis-active metalloproteinase activity at the NS2/NS3 cleavage site), NS3 (a serine proteinase/NTPase/RNA helicase), NS4A (serine proteinase accessory factor), NS4B, NS5A, and NS5B (RNA-dependent RNA polymerase).
With the exception of the 5′ non-translated RNA, there is substantial genetic heterogeneity among different stains of hepatitis C virus. Phylogenetic analyses have led to the classification of hepatitis C virus strains into a series of genetically distinct “genotypes,” each of which contains a group of genetically related viruses. The genetic distance between some of these genotypes is large enough to suggest that there may be biologically significant serotypic differences as well. There is little understanding of the extent to which infection with a virus of any one genotype might confer protection against viruses of a different genotype.
The currently available therapy of interferon in combination with ribavirin has poor response rate against most prevalent strains of HCV, genotype 1a and 1b. Establishment of selectable subgenomic replicon systems has advanced the study of HCV RNA replication. However, only replicons of genotype 1b strains are readily available, and extension of replicon systems to other genotypes has been largely unsuccessful. Considering the nature of high genetic variability of HCV, HCV replication systems derived from other genotypes will be very helpful in the effort of drug discovery. In support with this notion, chimeric replicons containing a genotype 1a polymerase in the background of a genotype 1b replicon were more resistant to interferon treatment in vitro than the replicon derived from a genotype 1b HCV. Extension of replicon system to other genotypes are also necessary to understand the mechanism of HCV RNA replication and the contribution of variable sequences in that process.
Recently two groups reported the generation of genotype 1a replication system using highly permissive sublines of Huh-7 cells. Blight et al. (J. Virol. 77, 3181-3190 (2003)) were able to select G418 resistant colonies supporting replication of genotype 1a derived subgenomic replicons in a hyper-permissive Huh7 subline, Huh-7.5, that was generated by curing an established G418-resistant replicon cell line of the cubgenomic Con1 replicon RNA that had been used to select it by treatment with interferon-alpha (Blight et al., J. Virol., 76, 13001-13014 (2002)). Sequence analysis of replicating HCV RNAs inside of such selected cell lines showed that the most common critical mutations were located at amino acid position 470 of NS3 (P1496L) within domain II of the NS3 helicase, and the NS5A mutation (S2204I). In other case, Grobler et al. (J. Biol. Chem., 278,16741-16746 (February 2003)), used a systematic mutational approach to reach the similar conclusion that both P1496L and S2204I combination was necessary to get genotype 1a replication in a highly permissive Huh-7 subline which was selected in an independent but similar way. However, genotype 1a RNAs with these two enhanced mutations does not undergo replication in the Huh-7 cell line, indicating limited usefulness of this system.